HIGH-LEVEL ACTIVITY OF THE MOUSE CCAAT ENHANCER-BINDING PROTEIN (C/EBP-ALPHA) GENE PROMOTER INVOLVES AUTOREGULATION AND SEVERAL UBIQUITOUS TRANSCRIPTION FACTORS
The promoter region of the mouse CCAAT-Enhancer Binding Protein (C/EBPalpha) gene is capable of directing high levels of expression of reporter constructs in various cell lines, albeit even in cells that do not express their endogenous C/EBPalpha gene. To understand the molecular mechanisms underlying this ubiquitous expression, we have characterized the promoter region of the mouse C/EBPalpha gene by a variety of in vitro and in vivo methods. We show that three sites related in sequence to USF, BTE and C/EBP binding sites and present in promoter region - 350/ + 3, are recognized by proteins from rat liver nuclear extracts. The sequence of the C/EBPalpha promoter that includes the USF binding site is also capable of forming stable complexes with purified Myc + Max heterodimers and mutation of this site drastically reduces transcription of C/EBPalpha promoter luciferase constructs both in liver and non liver cell lines. In addition, we identify three novel protein-binding sites two of which display similarity to NF-1 and a NFkappaB binding sites. The region located between nucleotides - 197 and - 178 forms several heat-stable complexes with liver nuclear proteins in vitro which are recognized mainly by antibodies specific for C/EBPalpha. Furthermore, transient expression of C/EBPalpha and to a lesser extent C/EBPbeta expression vectors, results in transactivation of a co-transfected C/EBPalpha promoter-luciferase reporter construct. These experiments support the notion that the C/EBPalpha gene is regulated by C/EBPalpha but other C/EBP-related proteins may also be involved.